Brassinosteroids: synthesis and biological activities

Brassinosteroids: synthesis and biological activities

Jana Oklestkova, Lucie Rárová, Miroslav Kvasnica & Miroslav Strnad

Phytochemistry Reviews Fundamentals and Perspectives of Natural Products Research ISSN 1568-7767 Volume 14 Number 6 Phytochem Rev (2015) 14:1053-1072 DOI 10.1007/s11101-015-9446-9

1 23

Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media Dordrecht. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.

1 23

Author's personal copy Phytochem Rev (2015) 14:1053–1072 DOI 10.1007/s11101-015-9446-9

Brassinosteroids: synthesis and biological activities Jana Oklestkova . Lucie Ra´rova´ . Miroslav Kvasnica . Miroslav Strnad

Received: 29 September 2015 / Accepted: 27 October 2015 / Published online: 31 October 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Brassinosteroids (BRs) are a relatively recently discovered group of phytohormones that are essential for normal plant growth and development. They participate in regulation of numerous vital physiological processes in plants, such as elongation, germination, photomorphogenesis, immunity and reproductive organ development. Structurally they are very similar to animal steroid hormones and include about 70 polyhydroxylated sterol derivatives. They are found at low levels in practically all plant organs. Recent studies have indicated that BRs have antiproliferative, anticancer, antiangiogenic, antiviral and antibacterial properties in animal cell systems, and thus have potential medical applications. Among others, BRs can inhibit replication of viruses in confluent human cell cultures, sometimes with high selectivity indexes, inducing cytotoxic effects in various types of cancer cells but not normal human cells. Thus, they include promising leads for developing potent new anticancer drugs. The aims of this article are to overview chemical characteristics, biological activities and the potential medical applications of natural BRs.

J. Oklestkova (&) L. Ra´rova´ M. Kvasnica M. Strnad Laboratory of Growth Regulators, Centre of the Region Hana´ for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR, Palacky´ University, Sˇlechtitelu˚ 27, 783 71 Olomouc, Czech Republic e-mail: [email protected]

Keywords Brassinosteroids Chemical synthesis Plant biological activity Antiproliferative activity Antiviral activity

Introduction Brassinosteroids (BRs) are a class of plant-specific steroid hormones characterized by polyhydroxylated sterol structures with significant growth-promoting activities (Clouse and Sasse 1998). They were initially reported at the start of the 1970s, when Mitchell et al. (1970, 1971) showed that ether extracts of Brassica napus (rape) pollen, designated ‘‘brassin’’, promote stem elongation and cell division in the bean internode assay. The first BR to be isolated, from 40 kg of beecollected rape pollen was named brassinolide (2) (Grove et al. 1979). All BRs have a 5a-cholestane skeleton, with functional variations due to differences in orientations of oxygenated functions on the skeleton (Bajguz and Tretyn 2003). BRs are distributed throughout the plant kingdom and have been isolated, to date, from 64 plant species including 53 angiosperms, one pteridophyte, one bryophyte and three algae (Bajguz 2011). They have been detected in all examined plant organs, including pollen, seeds, leaves, stems, roots, flowers and grains. They are also present in insect and crown galls. However, pollen and seeds are the richest sources of BRs, with contents in the 1–100 ng g-1 FW range, while shoots and leaves usually have much lower

123

Author's personal copy 1054

amounts, 0.01–0.1 ng g-1 FW (Bajguz 2011). BRs are involved in the regulation of many vital plant physiological activities, such as cell expansion, cell division, vegetative growth, reproduction, senescence, seed germination and stress tolerance (Clouse 2002; Bhardwaj et al. 2006; Krishna 2003). Recent studies have shown that BRs also have potent antiviral, antifungal, antiproliferative, antibacterial, neuroprotective and antiangiogenic activities in animal and human systems (Wachsman et al. 2000, 2002; Michelini et al. 2004, 2008; Malı´kova´ et al. 2008; Steigerova´ et al. 2010; Ra´rova´ et al. 2012). An array of brassinosteroids with variations in C-24 alkyl substituents are synthesized from campesterol, sitosterol, and cholesterol. All three sterols are converted to large numbers of metabolites in plant cells, but only a few of the metabolites have biological activity and biosynthetic pathways starting from campesterol seem to be the most important in planta (Hartmann 1998). Three major pathways of BR biosynthesis starting from this precursor are known. In two of these pathways the intermediate campestanol is converted via either an ‘‘early’’ or ‘‘late’’ C-6 oxidation route, in which C-6 oxidation occurs before and after introduction of C22 and C23 vicinal hydroxyls, respectively (Fujioka and Yokota 2003). These parallel pathways converge at castasterone (1), the immediate precursor of BL. Late C-6 oxidation is more prevalent in a number of species, including Arabidopsis and pea (Nomura et al. 2001). The third pathway, proposed by Ohnishi et al. (2006) following analysis of BR biosynthesis in Arabidopsis, involves direct (campestanol-independent) conversion of early C22-hydroxylated intermediates to 3-dehydro-6deoxoteasterone and 6-deoxotyhpasterol via C-23 hydroxylation. Proteins involved in BR signal transduction pathways have also been identified. In Arabidopsis BRs are perceived by the BR INTENSIVE1 (BRI1) plasma-membrane receptor kinase and activation of BRI1/BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) kinase complexes by transphosphorylation. Subsequently, BRASSINOSTEROID INSENSITIVE 2 (BIN2) kinase is dephosphorylated and inactivated, resulting in the accumulation of unphosphorylated BRASSINAZOLE RESISTANT (BZR) transcription factors in the nucleus (Kim and Wang 2010; Li and Chory 1997; Wang et al. 2001). In this review we summarize

123

Phytochem Rev (2015) 14:1053–1072

current knowledge about BRs’ chemistry and biological activities in plant and animal systems (Fig. 1).

Chemistry The first syntheses of BRs were described shortly after their isolation and structural determination, when Thompson et al. (1979) published syntheses of 24-epicastasterone (24-epiCS, 3) and 24-epibrassinolide (24-epiBl, 4). Starting from commercially available ergosterol (7), these BRs were respectively obtained after nine and 12 steps, as shown in Scheme 1. The most important reactions in this scheme are isomerization and formation of i-ergosterol (9), oxidation to i-sterone (10), reduction of the 7-double bond, formation of 2,3-olefin 14, cis-dihydroxylation, and Baeyer–Villiger oxidation. This is still a standard strategy for preparation of 24-epibrassinosteroids and few improvements have been published. However, McMorris and Patil (1993) improved synthesis of 24-epibrassinolide (4), reducing the number of reaction steps to seven. This was done by catalyzing isomerization of i-sterone (5) to 2,3olefin (14) using pyridinium hydrochloride and lithium bromide in one step, and performing the Baeyer–Villiger oxidation of 24-epicastasterone (3) using trifluoroperoxyacetic acid solution without prior acetylation of hydroxyl groups (in both cases with 80 % yield). If the starting material is brassicasterol 24-epibrassinolide (4) can be prepared in just five steps (Takatsuto and Ikekawa 1984). However, low availability and high price of brassicasterol limit its use as a starting material. 28-homobrassinolide (6) is prepared in a similar fashion to the synthesis of 24-epibrassinolide (4) already described. The starting material is usually commercially available stigmasterol (16), from which 28-homobrassinolide (6) can be obtained after six reaction steps (McMorris et al. 1994). Another synthetic approach, published by Sakakibara and Mori (1982), also starts from stigmasterol (16), but involves epoxidation of the 22,23-double bond in 17, followed by cleavage of the epoxide ring of 18 with hydrobromic acid and formation of bromohydrin (19). Acetylation of bromohydrin (19) and hydrolysis of bromide followed by saponification of tetraacetate (21) affords 24-homocastasterone (5) (Scheme 2).

Author's personal copy Phytochem Rev (2015) 14:1053–1072

1055

Fig. 1 Structures of representative natural brassinosteroids

24-Homobrassinolide (6) is then obtained by standard Baeyer–Villiger oxidation. The syntheses described thus far are straightforward, but a crucial reaction that significantly influences overall yields is osmium tetroxide-catalyzed asymmetric dihydroxylation of double bonds. While dihydroxylation of the double bond on the A-ring leads only to the 2a,3a-diol, dihydroxylation of the side chain double bond can afford both 22R,23R-diol (natural) and 22S,23S-diol (unnatural). The stereoselectivity of dihydroxylation mostly depends on the C-24 configuration and chiral ligand used in the reaction. It was proved that 9-O-(90 -phenanthryl)dihydroquinidine is so far the best chiral ligand for Sharpless dihydroxylation (OsO4 ? K3[Fe(CN)6] ? chiral ligand) of 22-ene steroids (Huang et al. 1993). The effect was most evident in case of (22E,24S)-24ethyl substituted side chain when the ratio of R,R- and S,S-diol was increased up to 8:1. Without chiral ligand this dihydroxylation gave only S,S-diol. In the case of (22E,24R)-24-methyl substituted side chain the same dihydroxylation procedure favors R,R-diol in a much higher ratio (13:1). Slow dihydroxylation of the side chain double bond can be accelerated by adding methansulfonamide (McMorris and Patil 1993). A completely different strategy has been developed for synthesis of the first discovered BR, brassinolide (2), and castasterone (1). This is mainly due to low

availability of starting material for the strategy used to synthesize 24-epibrassinolide (4) and 28-homobrassinolide (6). For example, crinosterol could potentially be used, but only analytical amounts that cannot be used for multistep procedures are available, although its synthesis has been described (Anastasia et al. 1983). Thus, efforts have mainly focused on preparation of a new side chain with a 24S-methyl group. There are many approaches for building a chain with the desired methyl group configuration. The first, published shortly after isolation of brassinolide (Fung and Siddall 1980), was based on the reaction of aldehyde 22 with lithium butyldimethyl-(E)-2,3dimethylbutenylalanate as a carbon–carbon formation reagent with 46 % yield. Subsequent epoxidation of olefin 23 and cleavage of epoxide 24 affords the desired side chain (Scheme 3). A similar procedure has also been described for preparation of 28-homobrassinolide (6) and dolicholide (Mori et al. 1984). Compound 23 can also be obtained from 20-formyl derivative 22 via three-step synthesis using 3-methyl-2-oxobutyl(triphenyl)arsonium bromide as a carbon–carbon bond formation reagent (Shen and Zhou 1990). Another synthetic strategy is based on formation of alkyne side chain (e.g. 23) as a key intermediate for further modification. The main reagent used for its synthesis is 3-methylbut-1-ynyl lithium, prepared

123


Phytochem Rev (2015) 14:1053–1072

Scheme 1 Synthesis of 24-epicastasterone (3) and 24-epibrassinolide (4) from ergosterol (7) (double arrows indicate improved and shortened synthesis)

in situ from 1,1-dibromo-3-methyl-but-1-ene. Ishiguro et al. (1980) published the first alkyne side chain synthesis using this reagent. However, such a synthesis of brassinolide (2) involves 22 steps and thus is very inefficient, in large part because reduction of the cyano group of 31 to methyl alone requires eight steps (Scheme 4). The same group also used this procedure for synthesizing typhasterol and several 28-norbrassinosteroids (Takatsuto et al. 1984). More efficient way was published few years later (Sakakibara and Mori 1983; Mori et al. 1984; Aburatani et al. 1985). The improvement that allowed significant reduction of reaction steps is based on opening of epoxide ring of 29 with trimethylaluminium directly to 24S methyl group (Scheme 4).

123

Although this strategy is more efficient in number of reaction steps, the overall yield is affected by nonstereoselectivity of the alkyne formation: both isomers of 22-hydroxy alkynes (27R and 27S) are obtained in 1:1 ratio. This problem was subsequently solved via stereoselective sulfenate-sulfoxide transformation (Zhou and Shen 1991). The mixture of both hydroxy alkynes (27S and 27R) can be hydrogenated to olefins 28S and 28R, which are then subjected to reaction with benzenesulfenylchloride to form sulfoxides 33S and 33R. Methylation with iodomethane in the presence of lithium diisopropylamide followed by cleavage of sulfoxide 34 with trimethyl phosphite affords the 22Rhydroxy-24-methyl olefin 35 and its 22S isomer in a 8.4:1 ratio with 53 % overall yield (Scheme 5).


1057

Scheme 2 Alternative synthesis of 28-homocastasterone (5) (undesired isomers omitted for clarity)

Scheme 3 Construction of brassinolide (2) with organoaluminum reagent

Another approach to build the side chain of brassinolide (2) is Julia-Lythgoe olefination with chiral sulfone (Sakakibara et al. 1982). Starting with aldehyde 26 and chiral (S)-2,3-dimethylbutyl-phenylsulfone this olefination can afford the side chain with desired 24S configuration (crinosterol side chain). Further reduction of sulfone 37 by sodium amalgam in methanol and ethyl acetate gives olefin 38, which can be oxidized by osmium tetroxide (Scheme 6). Efficient and stereocontrolled preparation of brassinolide (2) can be achieved by using of 3-isopropylbut-2-enolide

(McMorris et al. 1996). Its reaction with lithium diisopropylamide followed by addition of aldehyde 26 gave hydroxybutenolide 39. Such a lactone is hydrogenated and then reduced with lithium aluminum hydride to afford the side chain with desired configurations on all chiral carbons. Oxidation of primary alcohol 42 and further decarbonylation of aldehyde 43 with Wilkinson’s catalyst removes oxygen from position 28 (Scheme 7). The overall yield of this procedure is 32 %. A shorter and more efficient way of constructing the side chain involves a similar sequence

123


Phytochem Rev (2015) 14:1053–1072

Scheme 4 Two routes for constructing side chain 30 via alkyne

Scheme 5 Efficient construction of brassinolide side chain 30 via sulfoxide

of reactions but starts with 3,4-dimethylbut-2-enolide (Donaubauer et al. 1984). In this case, the key intermediate is 26-hydroxy derivative 47. The hydroxyl group is removed to obtain brassinolide side chain

123

by standard reactions for this purpose; mesylation and reduction of mesylate 49 with lithium aluminum hydride. The overall yield of this sequence is 70 % (Scheme 8).


1059

Scheme 6 Construction of the crinosterol side chain using Julia-Lythgoe olefination

Scheme 7 Construction of brassinolide side chain using 3-isopropylbut-2-enolide

Another option for controlling the side chain stereochemistry is to use furan-2-yl lithium as a reagent (Kametani et al. 1989; Honda et al. 1990; Tsubuki et al. 1992). Stereoselective hydride reduction of 52 and subsequent 1,4-addition of the 24-methyl group to the pyranone derivative 55 are the key reaction steps of this side chain (Scheme 9). Nucleophilic addition to aldehyde 26 has also been exploited, in the most recently published construction of the brassinolide side chain (Hurski et al. 2013, 2015), which can provide brassinolide (2) in a few steps via use of chiral thioacetal (Scheme 10). Selective reduction of 23-oxoderivate 60 (cryptolide side

chain) is afforded by presence of the bulky tbutyldimethylsilyloxy group on neighboring carbon. Moreover, this synthesis can also afford other BR biosynthetic precursors and metabolites (e.g. cathasterone 59 and cryptolide 58). The above mentioned approaches are landmark examples of BR syntheses during the last ca. 40 years. Many other published procedures have been patterned on them and others are probably awaiting publication, highlighting the intense interest among organic chemists working on plant hormones in finding simple procedures for synthesizing BRs and their natural analogues. Reasons for this interest are described below.

123


Scheme 8 Construction of brassinolide side chain using 2,3-dimethylbut-2-enolide

Scheme 9 Construction of brassinolide side chain using furan-2-yl lithium

123

Phytochem Rev (2015) 14:1053–1072


1061

Scheme 10 Construction of the brassinolide side chain using chiral thioacetal, including cryptolide 58 and cathasterone 59 side chain

Biological activities of BRs on plants BRs promote growth and developmental processes in all plant tissues and organs. At cellular levels, they regulate cell elongation, cell division and differentiation. At whole-plant levels, they participate in control of root and shoot development, seed germination and fertility (Kvasnica et al. 2014; Sasse 2003; Yang et al. 2011). BRs apparently co-ordinate and integrate diverse processes required for growth, partly via interactions with other hormones (Mu¨ssig 2005). In addition to their pivotal roles in plant growth and development, they appear to participate in mechanisms conferring tolerance to diverse environmental stresses, including high and low temperatures, drought, salinity, herbicidal injury, and pathogen attack (Krishna 2003). Effects of BRs on cell elongation and division Brassinosteroids participate in plant growth regulation by acting on both cell expansion and division. Genetic studies have confirmed that these processes are severely retarded in most BR-deficient and BR-insensitive mutants (Clouse et al. 1996). BR-deficient mutants display severe leaf phenotypic perturbations, including production of small, round leaves and short petioles (Szekeres et al. 1996; Li et al. 2001). Feeding these mutants with BRs restores the normal leaf size, by increasing both sizes and numbers of mesophyll

cells, implying that BRs promote leaf growth by enhancing both cell expansion and cell division (Nakaya et al. 2002). In rice the abaxial sclerenchyma cell number of lamina joints is closely related to leaf erectness and BR signaling tightly regulates their proliferation. Sun and co-workers identified a rice U-type cyclin CYC U4;1 that plays a positive role in promoting leaf erectness by controlling the abaxial sclerenchyma cell proliferation (Sun et al. 2015). A rice dwarf1 mutant, which lacks BR C-6 oxidase activity, exhibits abnormal organization and polar elongation of leaf and stem cells, leading to dramatic defects in development of many organs (Hong et al. 2002). Conversely, BR-insensitive (BRI1) mutants have larger and longer leaf blades, and longer petioles, than wild-type counterparts, due to increases in epidermal cell numbers, suggesting that BRs solely promote cell division during leaf growth (Gonzalez et al. 2010; Oh et al. 2011). However, Zhiponova et al. (2013) found that stimulating BR signaling and BR biosynthesis respectively promoted cell proliferation and cell differentiation. Hence, while enhancing BRI1 activity led to increased numbers of dividing cells (Gonzalez et al. 2010; Oh et al. 2011), continuous exposure to the hormone resulted in a lower proportion of mitotic cells and enhanced cell expansion (Zhiponova et al. 2013; Singh and Savaldi-Goldstein 2015) Early studies with Zinnia elegans L. mesophyll cell cultures showed that BRs also play an important role in promoting xylem differentiation (Yamamato et al.

123


Phytochem Rev (2015) 14:1053–1072

1997). Notably, the BR synthesis inhibitors uniconazole and brassinazole prevent xylem differentiation in Zinnia cell cultures, which can be restored by treatment with BL or 28-homoBL (Iwasaki and Shibaoka 1991). BL is essential for entry into the final stage of tracheary element differentiation, where secondary wall formation and cell death occur (Iwasaki and Shibaoka 1991).

appears to be regulated by BRs post-transcriptionally. Interestingly, BRs had similar effects on PIN4 accumulation in the columella, although Hacham et al. (2012) detected no apparent transcriptional perturbation of PIN 1, 3 and 7 genes in BR-deficient plants.

BRs regulate root growth

BRs have particularly strong growth-promoting effects in stems of seedlings and young plants (providing the basis for one of the most widely used BR bioassays, the second bean internode bioassay), mainly via promotion of expression of genes involved in cell elongation and wall extensibility (Horvath et al. 2003; Yang et al. 2011). Expression of the BR receptor BRI1 or a BR biosynthetic enzyme (CPD) in the epidermis, but not the vasculature, can rescue the dwarf phenotype of null mutants. BR signaling from the epidermis is not sufficient to establish normal vascular organization. Moreover, shoot growth is restricted when the epidermis is depleted of BRs and BRs act locally within a leaf (Savaldi-Goldstein et al. 2007). Ibanes et al. (2009) showed that BR signaling is required to set the number and arrangement of vascular bundles in Arabidopsis shoots, as BRdeficient and BR-insensitive mutants have fewer bundles, while BR signaling mutants have more than wild type stems. In Arabidopsis, BRL1 and BRL3 (but not BRL2) are functional genes involved in vascular tissue development (Cano-Delgado et al. 2004). Growth is also associated with increased carbohydrate demand for biosynthetic metabolism, and Goetz et al. (2000) provided evidence for a role of BR in carbohydrate allocation in hypocotyls, showing that expression of the tomato Lin6 gene is specifically induced by BR in the 10 mm section immediately below the hypocotyl hook. Furthermore, Tanaka et al. (2003) found that BR treatment can dramatically increase the length of hypocotyl cells in light-grown plants, while application of the BR-biosynthesis inhibitor brasinazole (Brz) reduces their average length. BRs act interactively with other plant hormones in the regulation of plant elongation, notably they have essentially additive effects with gibberellins (GA) and synergistic effects with auxin (IAA) on stem segment elongation (Unterholzner et al. 2015; Tong et al. 2014). More specifically, Katsumi (1985) showed that treating cucumber hypocotyl sections with 28-homoBl

Several recent studies have demonstrated the presence of BRs in maize and tomato roots (Kim et al. 2000; Yokota et al. 2001). The effects of BRs on root growth are strongly dependent on the BR concentration used: low (nM) concentrations can stimulate primary root growth, while higher concentrations (lM) can inhibit it (Haubrick and Assmann 2006). 24-EpiBl (4) reportedly has inhibitory effects on root formation in mung bean (Vigna radiata), wheat (Triticum aestivum), maize (Zea mays), tomato (Solanum lycopersicum) and Arabidopsis seedlings (Roddick and Ikekawa 1992; Clouse et al. 1993; Guan and Roddick 1988). Two recent studies on the role of BRs in root growth have shown that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem (GonzalezGarcia et al. 2011; Hacham et al. 2011; Vragovic et al. 2015; Chaiwanon and Wang 2015). More specifically, analyses of BRI1 loss of-function (bri1-116 null) and gain-of-function (bes1-D) Arabidopsis mutants by Gonzalez-Garcia et al. (2011) showed that balanced BR signaling is needed to maintain meristem size and overall root growth, and that BR signaling is required for cell cycle progression. Hacham et al. (2011) showed that the size of the root meristem is controlled by BRI1 activity in the epidermis and the signal from the epidermis is probably transmitted by a component that differs from BZR1 and BES1/BZR2. Further, BRs’ activity in regulation of root meristem size is integrated with the action of PIN auxin efflux carriers (Hacham et al. 2012), as bri1 mutants and plants treated with the BR biosynthesis inhibitor brassinazole, had lower than wild-type PIN2 levels. Moreover, restricting BRI1 activity in the epidermis restored PIN2 expression while BRI1 activity in the inner cells had weaker effects. In addition, levels of transcripts encoding endogenous PIN2 and transgenic PIN2-GFP slightly changed in response to short BR treatment, in opposite directions to PIN2 protein levels. Thus, PIN2

123

Effects of BRs on shoot growth


(6) followed by IAA resulted in synergistic enhancement of auxin-induced elongation, but when the order of treatment was reversed, 28-homoBl (6) was inactive, suggesting that BRs modulate stem tissues’ capacity to respond to IAA. Effects of various combinations of GAs, IAA and brassinolide (2) on the development and leaf anatomy of Tabebuia alba (Cham.) seedlings have also been evaluated (Ono et al. 2000). GA plus brassinolide (2) induced the highest stem and petiole growth rates of the tested combinations, accompanied by significant development of lateral buds, while Bl (2) application alone stimulated petiole growth but not stem growth. The effects of varying concentrations of IAA alone or in combination with Bl (2) or the cytokinin 6-benzylaminopurine (BAP), on ethylene production in Arabidopsis inflorescences have also been evaluated (Arteca and Arteca 2008). The cited authors found that treating inflorescences with Bl (2) alone had no effect on ethylene production. However, application of Bl (2) in combination with IAA increased ethylene production dramatically more than IAA alone, so co-application of these hormones had a synergistic effect on ethylene production. In addition, Bl (2) reportedly promotes stomatal closure and inhibits stomatal opening in Vicia faba L., by inhibiting inwardly rectifying K? currents of guard cell protoplasts in a similar manner to ABA (Haubrick et al. 2006). However, effects of applying Bl (2) and ABA together were not additive, suggesting that these two hormones may function in interacting pathways that regulate stomatal and guard cell physiology. Roles of BRs in reproductive organ development Very high BR levels are found in pollen and seeds, and extensive studies have demonstrated that BR plays a significant role in regulation of reproductive development in Arabidopsis (Chory et al. 1991; Li et al. 2010). On the female side, BRs influence numbers of ovule primordia and, thus, numbers of seeds produced (Huang et al. 2013), while dwarf BR Arabidopsis mutants display male sterility, due to abnormal tapetum development and reductions in pollen number (Ye et al. 2010). Conversely, exogenous application of 24-epiBL can stimulate Arabidopsis pollen germination and pollen tube growth rates, and increase final pollen tube lengths, in vitro (Vogler et al. 2014). BRs may also influence branching and flower formation through

1063

modulation of metabolic pathways and nutrient allocation patterns, then subsequently promote fertilization by stimulating filament and pollen growth (Mu¨ssig 2005). Numerous other factors are, of course, interactively influential. For example, Kesy et al. (2003) found that the short-day plant Pharbitis nil Choisy treated with Bl (2) and CS (1) formed fewer flowers than control plants, but the degree of inhibition depended on the concentration applied, the application method and length of the inductive dark period. Nevertheless, spraying BR at flowering generally leads to a significant increase in the production of various crops (Vriet et al. 2012). For example, Symons et al. (2006) found that exogenous application of 24-epiBl (4) and brassinazole respectively promoted and delayed ripening of grape (Vitis vinifera L.) fruits. Similarly, Vardhini and Rao (2002) found that applying 24-epiBl (4) and 28-homoBl (6) to tomato pericarp discs promoted ripening-associated changes, including increases in lycopene and carbohydrate contents, accompanied by increases in ethylene production and reductions in chlorophyll and ascorbic acid contents. Roles of BRs in seed development and germination Little is known about effects of BRs on seeds’ composition (protein, fat, mineral and carbohydrate contents). However, results of exogenous 24-epiBl (4) application on wheat and soybean (Glycine max) plants and seeds suggest that BRs significantly increase b-carotene and tocopherol contents, to varying degrees depending on the treatment method (Janeczko et al. 2009, 2010). Hayat and Ahmad (2003) found that 28-homoBL (6) treatment increased catalase, a-amylase and peroxidase levels of wheat seedlings. They also found that soaking Lens culinaris Medik seeds in 28-homoBl solutions resulted in seedlings with reduced root lengths and numbers of nodules, but higher than control nitrate reductase activities (Hayat and Ahmad 2003). In addition, analysis of seed development, and effects of BR on genes known to control various aspect of seed development, in BR-deficient (det2) and BR-insensitive (bri1-5) mutants by Jiang et al. (2013) demonstrated that BRs play crucial roles in regulation of the size, mass, and shape of Arabidopsis seeds. Their results indicate that BR increased seed size by affecting the integument, endosperm, and embryo development, and that BR-activated BZR1 directly

123


regulated several genes known to control seed size. They further showed that the seed shape was determined largely by BR signals from maternal tissues, while BRs produced by the embryo and endosperm seemed sufficient to increase seed size, providing evidence for a localized mode of BR action in seed development. BR applications also reportedly enhance germination of the seeds of certain parasitic angiosperms and cereals (Yamaguchi et al. 1987; Takeuchi et al. 1995). Leubner-Metzger (2001) extended these findings by showing that BR and the gibberellin GA4 and Bl both promote germination of Nicotiana tabacum L. cv. Havana 425 seeds, but by distinct signal transduction pathways. GAs and light release photodormancy via a common pathway, but BRs do not release photodormancy. Furthermore, induction of b GLU I, a molecular marker for endosperm rupture, is associated with the GA/light pathway, but not BR signaling. Instead, the BR pathway seems to promote rupture of non-dormant seeds’ endosperm by enhancing the growth potential of the embryo (Leubner-Metzger 2001). Additional insights have been provided by Nomura et al. (2004, 2007), who found that a severely BR-deficient mutant of pea, lk, produces irregularly shaped seeds, and that Bl and CS levels peaked, together with levels of transcripts of two BR C-6-oxidase genes (CYP85A1, CYP85A6), when pea seeds were rapidly growing. In early germination stages they also observed increases in levels of CS (but not Bl) in the growing tissues, accompanied by high levels of CYP85A1 and CYP85A6 transcripts. However, the level of 6-deoxocathasterone decreased while levels of downstream intermediates increased. These results suggest that during germination and early growth stages CS plays a significant role, while 6-deoxocathasterone is the major BR for storage and utilization (Nomura et al. 2004). The transgenic rice plants expressing maize, rice or Arabidopsis genes encoding sterol C-22 hydroxylases that control BR hormone levels using a promoter that is active in only the stems, leaves and roots were created by Wu and co-workers. These plants produced more tillers and more seed than wild-type plants. The phenotypic changes brought about 15–44 % increases in grain yield per plant relative to wild-types in greenhouse and field trial (Wu et al. 2008). Roles of BRs in stress tolerance BRs play important roles in tolerance to diverse biotic and abiotic stresses, including drought, salt,

123

Phytochem Rev (2015) 14:1053–1072

heat, cold, hypoxia, pesticides, heavy metals and disease (Bajguz and Hayat 2009). Drought, salinity and freeze-induced dehydration constitute direct osmotic stresses, and thus share some effects with chilling and hypoxia, which can indirectly induce osmotic stress by impairing water uptake and increasing water losses. BR can reportedly increase Indian mustard plants’ tolerance of water stress, as both foliar sprays of 28-homoBl (6) and exposure to drought can raise their levels of antioxidant systems (Fariduddin et al. 2009). Application of 28-HomoBl (6) can also stimulate growth of wheat (Triticum aestivum L.) under drought stress, through increasing relative water contents, nitrate reductase activity, chlorophyll contents and photosynthesis rates (Sairam 1994), and these effects are stronger in drought-tolerant varieties. Further studies demonstrated that 24-epiBl (4) confers cucumber plants with hypoxia tolerance, probably by enhancing sugar supplies to hypoxic roots and shifting hypoxic metabolism from lactate fermentation to alcohol fermentation (Kang et al. 2009). Furthermore, treatment with Bl (2) enhances increases in superoxide dismutase, catalase, ascorbate peroxidase, ascorbic acid and carotenoid contents in maize seedlings subjected to water stress (Li et al. 1998). Similarly, the Arabidopsis det2 mutant is insensitive to the negative growth effects of very low oxygen concentrations, and its enhanced oxidative stress resistance has been associated with constitutive increases in superoxide dismutase activity and catalase transcript levels, suggesting that long-term BR deficiency results in a constant in vivo physiological stress in this mutant (Cao et al. 2005). Treating tomato and rape seedlings with 24-epiBl (4) increases thermotolerance, which is associated with higher synthesis of heat shock proteins (HSPs) and expression of mitochondrial small HSPs in the leaves (Dhaubhadel et al. 2002; Kagale et al. 2007). Finally, cultures of Chlorella vulgaris BEIJERNICK treated with BRs and heavy metals show lower bioaccumulation of the heavy metals zinc, cadmium, lead and copper than cultures treated with the metals alone (with effects declining in the presented order of metals). Hence, BRs reduce the impact of heavy metals stress on growth of these cultures, by decreasing chlorophyll, sugar and protein losses and increasing synthesis of phytochelatins (Bajguz and Hayat 2009; Bajguz 2000, 2002).


1065

Roles of BRs in plant immunity

Antiviral effects of BRs

Exogenously applied BRs can induce resistance to a broad spectrum of pathogens. For example, Bl (2) can protect rice against blast and bacterial blight diseases, and enhance resistance of tobacco to the bacterial pathogen Pseudomonas syringe and fungal pathogen Oidium (Nakashita et al. 2003). Similarly, Kripach et al. (2000) found that cultivation in medium containing Bl (2), 24-epiBl (4) and 28-homoBl (6) increased potato cuttings’ resistance to viral infection during all developmental stages. 24-EpiBl can also protect barley against Fusarium diseases: in the absence of BR, 31 % of spikelets inoculated with F. culmorum by Shahin et al. (2013) displayed typical Fusarium head blight bleaching symptoms. In contrast, just 4 % of spikelets treated with 24-epiBl showed such symptoms and they had substantially lower losses in grain number and weight. Other authors have shown that Bl treatment can increase Cucumber mosaic virus resistance in Arabidopsis by enhancing antioxidative enzyme activities and expression of defense-associated genes (Zhang et al. 2015). Furthermore, numerous studies have indicated that BR signaling components play essential roles in innate immunity, particularly BAK1, which besides participating in BR signaling is involved in regulation of microbe-induced cell death and interacts with various pattern recognition receptors (Kemmerling et al. 2007; Albrecht et al. 2012; Belkhadir et al. 2012).

Antiviral activities of various natural BRs have been reported recently, including in vitro activities of 28-homoCS (5) and 28-homoBl (6) against herpes virus, measles and arenaviruses (Wachsman et al. 2000, 2002). Brassinolide (2) and 28-homoCS (5) also have broad antiviral activities at lM levels, against both RNA and DNA viruses, including poliovirus, HSV-1 and HSV-2, measles virus, vesicular stomatitis virus (and the arenaviruses Junin, Tacaribe and Pichinde), mediated by interference with virus protein synthesis and maturation of viral particles (Wachsman and Castilla 2012). Several series of synthetic BR analogues have also been tested for antiherpetic and other antiviral activities, and ranked in terms of inhibitory effects on viral replication in confluent, non-growing Vero cell cultures (Michelini et al. 2004; Ramı´rez et al. 2000; Wachsman et al. 2004). Many of the BR derivatives were active against different viruses. The compounds were also usually cytotoxic, with varying selectivity indexes, and only a few exhibited medium cytotoxicity towards Vero cells (IC50 values between 20 and 60 lM).

Biological activities of BRs in human cells Roles of BRs in plant cells and plants have been intensively examined. Much less is known about their effects on human cells. However, as reviewed in the following sections, various studies have shown that natural BRs have potentially valuable activities for medical applications, including antiviral (Wachsman et al. 2000; Wachsman and Castilla 2012), immunomodulatory and neuroprotecting activity (Michelini et al. 2008, 2013), antioxidant and neuroprotective activities in a mammalian neuronal cell line (Carange et al. 2011), and antiproliferative effects in animal cells in vitro (Franek et al. 2003; Malı´kova´ et al. 2008; Obakan et al. 2014a, b; Ra´rova´ et al. 2012; Steigerova´ et al. 2010, 2012; Wu and Lou 2007).

Antiproliferative activities of BRs To date, more than 70 anticancer molecules have been derived from natural products (Newman and Cragg 2007), and many other natural products have been identified as potential sources of new anticancer drugs. The first published indications of relevant activities were stimulatory effects of 24-epiBl (4) on cultured hybridoma mouse cells, including increases in mitochondrial membrane potential and proportions of cells in the G0/G1 phase, and reductions in intracellular antibody levels and proportions of cells in S phase (Franek et al. 2003). Subsequently, the natural BRs 24-epiBl (4) and 28-homoCS (5) were shown to have inhibitory effects on the growth and viability of various cancer cell lines (Malı´kova´ et al. 2008). Promisingly, these BR substances affected the viability of human cancer cell lines (including CEM T-lymphoblastic leukemia, MCF7 breast carcinoma, A549 lung carcinoma, K562 chronic myeloid leukemia, RPMI 8226 multiple myeloma, HeLa cervical carcinoma, G361 malignant melanoma and HOS osteosarcoma lines) without affecting growth of normal human cells (BJ fibroblasts). Treatments with

123


28-homoCS (5) and 24-epiBl (4) resulted in potent, dose-dependent reductions in the viability of CEM and RPMI 8226 cells, with IC50 levels ranging from 13 to 50 lM. 28-HomoCS (5) was the most active compound against CEM cells (IC50 13 lM), inducing approximately three times stronger responses than 28-homoBl (6), indicating that transformation of 6-oxo-7-oxalactone to 6-oxo functionality substantially increases the growth inhibitory activity of BRs. The presence of a 24R side chain strongly reduces BR cytotoxicity. The 24R side chain in 24-epicastasterone also reduced anticancer activity compared to castasterone. 28-HomoCS (5) and 28-homoBl (6) have an ethyl group in their side chains at C24 and are more effective than the corresponding analogues with C24 methyl groups. Since b-ecdysone, which contains 2b,3b,22a–functionality, showed no detectable activity, the most important groups for strong cytotoxic BR effect could be a 3a-hydroxy group, 2a,3a-vicinal diol or 3a,4a-vicinal diol (Malı´kova´ et al. 2008). The cited authors also found that natural BRs had varying effects on estrogen- and androgen-sensitive and insensitive breast and prostate cancer cell lines, but they were more potent towards the hormone-sensitive cell lines, possibly due to their modulation of steroid receptors. Flow cytometry-based analysis revealed that the BRs can disturb the cell cycle in breast and prostate cancer cell lines. More specifically, 28-homoCS (5) and 24-epiBl (4) blocked the cell cycle in the G1 phase with concomitant reductions in percentages of cells in the S phase (Malı´kova´ et al. 2008). These are typical growth inhibitory effects of antiestrogens on MCF7 cells (Parl 2000). More recently, Hamdy et al. (2009) isolated a new natural BR-related metabolite, 3-keto22-epi-28-norcastasterone, from Cystoseira myrica that exhibited anticancer activity against human liver (HAPG-2) and colon cancer (HCT116) cells with IC50 values of 5.63 and 1.16 lM, respectively. Furthermore, 24-epiBl (4) can induce apoptosis by activating a polyamine catabolic pathway in LNCaP and DU145 prostate cancer cells, thereby inducing accumulation of cytotoxic hydrogen peroxide and aldehydes, and LNCaP cells expressing androgen receptor (AR) are more sensitive than cells lacking functional AR (Obakan et al. 2014a). The same authors also showed that 24-epiBl (4) can induce mitochondriamediated and caspase-dependent apoptosis, regardless

123

Phytochem Rev (2015) 14:1053–1072

of p53 expression, in androgen-insensitive PC3 prostate cancer cells. Biosynthetic precursors of natural 24-norbrassinolide phytohormones containing low-polarity groups in the cyclic moiety of the molecule also have antiproliferative and apoptosis-inducing activities towards LNCaP human prostate adenocarcinoma cells, mediated by blockage of the cell cycle (Khripach et al. 2012). BRs’ effects in wound healing and angiogenesis The growth of new blood vessels in animals, angiogenesis, is essential for organ growth, growth of solid tumors, and establishment of metastasis (Folkman 1974), to meet the organs’ or tumors’ oxygen and nutrient supply requirements. The most important components of angiogenic tissues, the endothelial cells, could be targets for antiangiogenic therapy because they are not transformed, and easily accessible to antiangiogenic agents. Furthermore, tumor angiogenesis could be potentially targeted by novel generations of drugs (of natural or synthetic origin) that inhibit proteolytic enzymes which break down the extracellular matrix surrounding existing capillaries, and/or inhibit endothelial cell proliferation and migration, and/or enhance apoptosis of tumors’ endothelial cells (Kesisis et al. 2007; Bhat and Singh 2008). Such candidate leads include various steroids with known antiangiogenic activity, among others 2-methoxyestradiol, progestin, medroxyprogesterone acetate, and glucocorticoids such as dexamethasone and cortisone (Pietras and Weinberg 2005). Several natural BRs also have effects on endothelial cells, including potential antiangiogenic activity (Ra´rova´ et al. 2012). Furthermore, two natural BRs, 24-epiBl (4) and 28-homoCS (5), reportedly inhibit growth of Human Microvascular Endothelial Cells (HMEC-1) in a dose-dependent manner, and reduce migration of Human Umbilical Vein Endothelial Cells (HUVEC). Treatment with these agents slightly reduced numbers of tubes that formed, as well as numbers of nodes, one of the parameters usually used to assess antiangiogenic activity. This antiangiogenic activity of BRs, along with their antiproliferative activity, suggests that these plant hormones and new synthetic derivatives with stronger antiangiogenic activities are potentially important leads for new anticancer drugs (Ra´rova´ et al. 2012).


BR effects on neuronal cells Oxidative stress and apoptosis are frequently implicated in the neuronal cell damage associated with various neurodegenerative disorders, such as Parkinson’s disease. BRs are known to promote stress tolerance in plants via modulation of the antioxidative enzyme cascade. Their antioxidative effects on mammalian neuronal cells have not yet been examined in detail. However, Carange et al. (2011) analyzed the ability of 24-epiBl to protect PC12 neuronal cells from oxidative stress induced by 1-methyl-4-phenylpyridinium, MPP(?), and consequent apoptosis in dopaminergic neurons. They found that 24-epiBl reduced levels of intracellular reactive oxygen species, and modulated superoxide dismutase, catalase, and glutathione peroxidase activities. The observed antioxidative properties of 24-epiBl led to the inhibition of MPP(?)-induced apoptosis by reducing DNA fragmentation, the Bax/Bcl-2 protein ratio and caspase-3 cleavage. This was the first demonstration of the potent antioxidant and neuroprotective effects of 24-epiBl in a mammalian neuronal cell line (Carange et al. 2011). Molecular mechanism of BR action in human cells Signaling in plants by BRs, and the resulting genomic responses, are initiated by a BR molecule binding to a receptor kinase, brassinosteroid-insensitive 1 (BRI1), localized in the plasma membrane (Kim and Wang 2010). However, there are differences between steroids’ actions in animal and plant cells. In animal models, lipophilic steroids bind to steroid receptors located in either the cytosol or nucleus that diffuse through the plasma membrane. Ligand binding induces a conformational change and dimerization with another receptor that allows the ligand/receptor complex to bind to target DNA sequences and directly modify gene expression over hours or even days (Losel and Wehling 2003). Because of the similarity between BRs and human steroids, we have studied interactions of BRs with human steroid receptors using reporter assays and a competitive binding assay. Reporter assays showed that 24-epiBl (4) is a weak antagonist of estrogenreceptor-a (ER-a). However, we found no evidence that 24-epiBl (4) and 28-homoCS (5) directly bind to ER-a and ER-b in competitive binding assays.

1067

24-EpiBl and 28-homoCS were also found to modulate levels of PR in a dose–response manner by western blotting, although we did not observe any interaction in the cellular reporter assay for PR response. Treatment with 24-epiBl increased protein levels of PR, while 28-homoCS led to downregulation of PR protein expression using western blotting. It seems possible that there are other mode of actions, both steroid receptor-dependent and independent, similarly to what is considered for 2-methoxyestradiol, which binds to estrogen receptors. Furthermore, the action mechanism of the antiangiogenic response is probably unrelated to the receptor pathway (Ra´rova´ et al. 2012). BR treatment has been shown to affect ER-a and ER-b localization patterns in MCF7 breast adenocarcinoma cells by Steigerova´ et al. (2010), who observed strong and uniform ER-a immuno-nuclear labelling in untreated MCF7 cells, and cytoplasmic speckles of ER-a immunofluorescence in MCF7 cells treated with 28-homoCS (5) or 24-epiBl (4). In contrast to ER-a, ER-b was predominantly found in the cytoplasm of untreated MCF7 cells. However, ER-b was notably relocated to the nuclei after 28-homoCS (5) treatment, whereas it was predominantly present at the periphery of the nuclei, in 24-epiBl (4)-treated cells. These changes were accompanied by down-regulation of the ERs following BR treatment (Steigerova´ et al. 2010). As mentioned above, these natural BRs inhibited cell growth and blocked the cell cycle at G1 phase, with concomitant reductions in percentages of cells in the S phase. Concomitant reductions in expression of key cycling proteins—including cyclin-dependent kinases (CDKs) 2/4/6 and cyclins D1 and E—and pRb phosphorylation were also detected during cell cycle arrest, accompanied by up-regulation of the cyclindependent kinase inhibitors p21Waf1/Cip1 and p27Kip1, which inhibit cyclin/CDK complexes. BRs also induced apoptosis in both estrogen-sensitive and estrogen-insensitive breast cancer cell lines, by distinct modulation of the expression of apoptosis-related proteins (Steigerova´ et al. 2010). Induction of apoptosis after the treatment with BRs was confirmed by TUNEL staining and double staining with propidium iodide and acridine orange in both MCF7 and MDAMB-468 breast cancer cell lines, but the changes in expression profiles of apoptosis-related proteins triggered by the BRs differed between the lines. In MCF7 cells, the BR treatments induced expression of the

123


antiapoptotic Bcl-2 and Bcl-XL proteins, and slightly affected levels of p53 and MDM-2 proteins. In MDAMB-468 cells cleavage of caspase-3 into fragments (part of the apoptotic cascade) 24 h after the BR treatment was followed by G1 phase arrest and increases in the subG1 fraction, representing apoptotic bodies. The mechanism of action of BRs in human cells is still largely unknown, but it seems that BRs may interact with one or more of the numerous steroidbinding proteins. Moreover, BRs could also induce multiple effects, both steroid receptor-dependent and independent (Steigerova´ et al. 2010). Both BRs also inhibited cell growth, induced G1 blocks and reduced expression of cyclin D1, CDK4/6 and pRb in LNCaP androgen-sensitive prostate cancer cells, while in androgen-insensitive DU-145 prostate cancer cells they increased proportions of cells in the G2/M phase, and down-regulated cyclins A and B1. Changes in AR localization patterns in LNCaP cells treated with BRs were also detected immunofluorescently. Apoptosis caused by BRs was detected in both prostate cancer cell lines, but (as observed following BR treatment of breast cancer cells) changes in their expression profiles of apoptosis-related proteins differed (Steigerova´ et al. 2012). Effects of 24-epiBl (4) in androgen-sensitive LNCaP prostate cancer cells, androgen-insensitive DU145 prostate cancer cells and PNT1a normal prostate epithelial cells were also examined to probe possible mechanisms of apoptotic cell death and related changes in polyamine biosynthetic and catabolic pathways. 24-EpiBl (4) decreased intracellular polyamine levels and significantly downregulated ornithine decarboxylase in both prostate cancer cell lines, and also modulated antizyme and antizyme inhibitor expression levels in LNCaP cells. In both cell lines, it also up-regulated expression of the catabolic enzymes spermidine/spermine N1-acetyltransferase and polyamine oxidase. Adding specific inhibitors of these enzymes and co-treatment with 24-epiBl (4) for 24 h decreased formation of cleaved PARP fragments in LNCaP cells (Obakan et al. 2014a). In conclusion, BRs seem to have extremely potent activities indicating that they may be valuable natural leads for novel anticancer drugs. Acknowledgments This work was funded by the Ministry of Education, Youth and Sports of the Czech Republic—NPU I program with project LO1204.

123

Phytochem Rev (2015) 14:1053–1072

References Aburatani M, Takeuchi T, Mori K (1985) Structural revision of the acetal intermediates in brassinolide synthesis. Agric Biol Chem 49:3557–3562 Albrecht C, Boutrot F, Segonzac C, Schwessinger B et al (2012) Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. PNAS 109:303–308 Anastasia M, Allevi P, Ciuffreda P, Fiecchi A (1983) Stereoselective synthesis of crinosterol [(22E,24S)-Ergosta-5,22dien-3b-ol]. J Chem Soc Perkin Trans 1:2365–2367 Arteca RN, Arteca JM (2008) Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants. J Exp Bot 59(11):3019–3026 Bajguz A (2000) Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chlorea vulgaris. Plant Physiol Biochem 38:209–2015 Bajguz A (2002) Brassinosteroids and lead as stimulators of phytochelatins synthesis in Chlorella vulgaris. J Plant Physiol 159:321–324 Bajguz A (2011) Brassinosteroids—occurence and chemical structures in plants. In: Hayat S, Ahmad A (eds) Brassinosteroids a class of plant hormone. Springer, London, pp 1–29 Bajguz A, Hayat S (2009) Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiol Biochem 47:1–8 Bajguz A, Tretyn A (2003) The chemical characteristic and distribution of brassinosteroids in plants. Phytochem 62:1027–1046 Belkhadir Y, Jaillais Y, Balsemao-Pires E, Dangl JL, Chory J (2012) Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. PNAS 109:297–302 Bhardwaj R, Arora HK, Nagar PK, Thukral AK (2006) Brassinosteroids-A novel group of plant hormones. In: Trivedi PC (ed) Plant molecular physiology-current scenario and future projections. Aaviskar Publisher, Jaipur, pp 58–84 Bhat TA, Singh RP (2008) Tumor angiogenesis—a potential target in cancer chemoprevention. Food Chem Toxicol 46(4):1334–1345 Cano-Delgado A, Yin Y, Yu C, Vafeados D et al (2004) BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131:5341–5351 Cao S, Xu Q, Cao Y, Qian K et al (2005) Loss-of-function mutations in DET2 gene lead to an enhanced resistance to oxidative stress in Arabidopsis. Physiol Plant 123:57–66 Carange J, Longpre´ F, Daoust B, Martinoli MG (2011) 24-Epibrassinolide, a phytosterol from the brassinosteroid family, protects dopaminergic cells against MPP-induced oxidative stress and apoptosis. J Toxicol 2011:392859 Chaiwanon J, Wang ZY (2015) Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots. Curr Biol 25(8):1031–1042 Chory J, Nagpal P, Peto CA (1991) Phenotypic and genetic analysis of det2, a new mutant that affects light-regulated

Author's personal copy Phytochem Rev (2015) 14:1053–1072 seedling development in Arabidopsis. Plant Cell 3:445–459 Clouse SD (2002) Brassinosteroid signal transduction: clarifying the pathway from ligand perception to gene expression. Mol Cell 10:973–982 Clouse SD, Sasse JM (1998) Brassinosteriods: essential regulators of plant growth and development. Ann Rev Plant Physiol Plant Mol Biol 49:427–451 Clouse SD, Hall AF, Langford M et al (1993) Physiological and molecular effects of brassinosteroids on Arabidopsis Thaliana. J Plant Growt Regul. 12:61–66 Clouse SD, Langford M, McMorris T (1996) A brassinosteroidinsensitive mutant in Arabidopsis Thaliana exhibits multiple defects in growth and development. Plant Physiol 111:671–678 Dhaubhadel S, Browning KS, Gallie DR, Krishna P (2002) Brassinosteroid functions to protect the translational machinery and heat-shock protein synthesis following thermal stress. Plant J. 29:681–691 Donaubauer JR, Greaves AM, McMorris TC (1984) A novel synthesis of brassinolide. J Org Chem 49:2834–2837 Fariduddin Q, Khanam S, Hasan S, Ali B et al (2009) Effect of 28-homobrassinolide on the drought stress-induced changes in photosynthesis and antioxidant system of Brassica juncea L. Acta Physiol Plant. 31:889–897 Folkman J (1974) Tumor angiogenesis. Adv Cancer Res 19:331–358 Franek F, Eckschlager T, Kohout L (2003) 24-Epibrassinolide at subnanomolar concentrations modulates growth and production characteristics of a mouse hybridoma. Collect Czech Chem Commun 68:2190–2200 Fujioka S, Yokota T (2003) Biosynthesis and metabolism of brassinosteroids. Ann Rev Plant Biol 54:137–164 Fung S, Siddall JB (1980) Stereoselective synthesis of brassinolide: a plant growth promoting steroidal lactone. J Am Chem Soc 102:6580–6581 Goetz M, Godt DE, Roitsch T (2000) Tissue-specific induction of the mRNA for an extracelluar invertase isoenzyme of tomato by brassinosteroids suggest a role for steroid hormones in assimilate partitioning. Plant J 22:515–522 Gonzalez N, De Bodt S, Sulpice R et al (2010) Increased leaf size: different means to an end. Plant Phys 153:1261–1279 Gonzalez-Garcia MP, Vilarrasa-Blasi J, Thiponova M, Divol F, Mora-Garcia S, Russinova E, Cano-Delgado A (2011) Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots. Development 138:849–859 Grove MD, Spencer FG, Rohwedder WK, Mandava NB, Worley JF, Wrthen JD Jr, Cook JC Jr (1979) Brassinolide, a plant growth promoting steroid isolated from Brassica napus pollen. Nature 281:216–217 Guan M, Roddick J (1988) Epibrassinolide inhibition of development of excised, adventitious and intact roots of tomato: comparison with the effects of steroidal estrogens. Physiol Plant 74:720–726 Hacham Y, Holland N, Butterfield C, Chory J, Savaldi- Goldstein S (2011) Brassinosteroid perception in the epidermis controls root meristem size. Development 138:839–848 Hacham Y, Sela A, Friedlander L, Savaldi-Goldstein S (2012) BRI1 activity in the root meristem involves post-

1069 transcriptional regulation of PIN auxin efflux carriers. Plant Signal Behav 7:1–3 Hamdy AH, Aboutabl EA, Sameer S, Hussein AA, Dıáz-Marrero AR, Darias J, Cueto M (2009) 3-Keto-22-epi-28-norcathasterone, a brassinosteroid-related metabolite from Cystoseira myrica. Steroids 74(12):927–930 Hartmann MA (1998) Plant sterols and the membrane environment. Trends Plant Sci 3:170–175 Haubrick L, Assmann S (2006) Brassinosteroids and plant function: some clues, more puzzles. Plant Cell Env. 29: 446–457 Haubrick LL, Torsethaugen G, Assmann SM (2006) Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts. Phys Plantarum 128:134–143 Hayat S, Ahmad A (2003) Soaking seeds of Lens culinaris with 28-homobrassinolide increased nitrate reductase activity and grain yield in the field in India. Ann Appl Biol 143: 121–124 Honda T, Keino K, Tsubuki M (1990) A concise stereoselective synthesis of castasterone. J Chem Soc Chem Commun 8:650–652 Hong Z, Ueguchi-Tanaka M, Shimizu-Sato S, Inukai Y et al (2002) Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6-oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. Plant J. 32:495–508 Horvath DP, Schaffer R, West M, Wisman E (2003) Arabidopsis microarrays identify conserved and differentially expressed genes involved in shoot growth and development from distantly related plant species. Plant J 34:125–134 Huang LF, Zhou WS, Sun LQ, Pan XF (1993) Studies on steroidal plant-growth regulators. Part 29. Osmium tetroxidecatalysed asymmetric dihydroxylation of the (22E,24R)and the (22E,24S)-24-alkyl steroidal unsaturated side chain. J Chem Soc Perkin Trans 1:1683–1686 Huang HY, Jiang WB, Hu YW, Wu P, Zhu JY, Liang WQ, Wang ZY, Lin WH (2013) BR signal influences Arabidopsis ovule and seed number through regulating related genes expression by BZR1. Mol Plant 6(2):456–469 Hurski A, Zhabinskii V, Khripach V (2013) A short convergent synthesis of the side chains of brassinolide, cathasterone, and cryptolide. Tetrahedron Lett 54:584–586 Hurski AL, Ermolovich YV, Zhabinskii VN, Khripach VA (2015) The development and use of a general route to brassinolide, its biosynthetic precursors, metabolites and analogues. Org Biomol Chem 13:1446–1452 Ibanes M, Fabregas N, Chory J, Cano-Delgado AI (2009) Brassinosteroid signaling and auxin transport are required to establish the periodic pattern of Arabidopsis shoot vascular bundles. Proc Natl Acad Sci USA 106:13612–13617 Ishiguro M, Takatsuto S, Morisaki M, Ikekawa N (1980) Synthesis of brassinolide, a steroidal lactone with plant-growth promoting activity. J Chem Soc Chem Commun 962–964 Iwasaki T, Shibaoka H (1991) Brassinosteroids act as regulators of tracheary-element differentiation in isolated Zinnia mesophyll cell. Plant Cell Physiol 32:1007–1014 Janeczko A, Biesaga Koscielniak J, Dziurka M (2009) 24-Epibrassinolide modifies seed composition in soybean, oilseed rape and wheat. Seed Sci Technol. 37:625–639

123

Author's personal copy 1070 Janeczko A, Biesaga-Koscielniak J, Oklestkova J et al (2010) Role of 24-epibrassinolide in wheat production: physiological effects and uptake. J Agron Crop Sci 196:311–321 Jiang WB, Huang HY, Hu YW, Zhu SW et al (2013) Brassinosteroids regulates seed size and shape in Arabidopsis. Plant Phys. 162:1965–1977 Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007) Brassinosteroid confers tolerance in Arabidopsis Thaliana and Brassica napus to a range of abiotic stresses. Planta 225:353–364 Kametani T, Keino K, Kigawa M, Tsubuki M, Honda T (1989) Stereocontroled synthesis of the brassinolide side chain via pyranone derivative. Tetrahedron Lett 30:3141–3142 Kang Y, Guo S, Li J, Duan J (2009) Effect of root 24-epibrassinolide on carbohydrate status and fermentative enzyme activities in cucumber (Cucumis sativum L.) seedlings under hypoxia. Plant Growth Regul 57: 259–269 Katsumi M (1985) Interaction of a brassinosteroid with IAA and GA3 in the elongation of cucumber hypocotyl sections. Plant Cell Physiol 26:615–626 Kemmerling B, Schwedt A, Rodriguez P, Mazzotta S et al (2007) The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control. Curr Biol 17:1116–1122 Kesisis G, Broxterman H, Giaccone G (2007) Angiogenesis inhibitors. drug selectivity and target specificity. Curr Pharm Des 13(27):2795–2809 Kesy J, Trzaskalsky A, Galoh E, Kopcewicz J (2003) Inhibitory effect of brassinosetroids on the flowering of the short-day plant Pharbitis nil. Biol Plant 47:597–600 Khripach VA, Zhabinskii V, Ermolovich Y, Gulyakevich O, Mekhtiev A, Karalkin P (2012) Synthesis and biological activity of the probable biosynthetic precursors of 24(1)norbrassinolide. Russ J Bioorg Chem 38:438–446 Kim TW, Wang ZY (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Ann Rev Plant Biol 61:681–704 Kim SK, Chang SC, Lee EJ, Chung WS et al (2000) Involvement of brassinosteroids in the gravitropic response of primary root of maize. Plant Physiol 123:997–1004 Kripach V, Zhabinskii V, De Groot A (2000) Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI century. Ann Bot 86:441–447 Krishna P (2003) Brassinosteriods- mediated stress responses. J Plant Growth Regul 22:289–297 Kvasnica M, Oklestkova J, Bazgier V, Rarova L, Berka K, Strnad M (2014) Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain. Steroids 85:58–64 Leubner-Metzger G (2001) Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways. Planta 213(5):758–763 Li J, Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929–938 Li L, Van Staden J, Ja¨ger AK (1998) Effects of plant growth regulators on the antioxidant system in seedlings of two maize cultivars subjected to water stress. Plant Growth Regul 25:81–87

123

Phytochem Rev (2015) 14:1053–1072 Li J, Nam KH, Vafeados D, Chory J (2001) BIN2, a new brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol 127:14–22 Li J, Li Y, Chen S, An L (2010) Involvement of brassinosteroid signals in the floral-induction network of Arabidopsis. J Exp Bot 61:4221–4230 Losel R, Wehling M (2003) Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol 4(1):46–56 Malı´kova´ J, Swaczynova´ J, Kola´rˇ Z, Strnad M (2008) Anticancer and antiproliferative activity of natural brassinosteroids. Phytochemistry 69(2):418–426 Mandava N, Mitchell JW (1971) New plant hormones: chemical and biological investigations. Indian Agr 15:19–31 McMorris TC, Patil PA (1993) Improved synthesis of 24-epibrassinolide from ergosterol. J Org Chem 58:2338–2339 McMorris TC, Patil PA, Chavez RG, Baker ME, Clouse SD (1994) Synthesis and biological activity of 24-homobrassinolide and analogues. Phytochemistry 36:585–589 McMorris TC, Chavez RG, Patil PA (1996) Improved synthesis of brassinolide. J Chem Soc Perkin Trans 1:295–302 Michelini FM, Ramirez JA, Berra A, Galagovsky LR, Alche LE (2004) In vitro and in vivo antiherpetic activity of three new synthetic brassinosteroid analogues. Steroids 69: 713–720 Michelini FM, Berra A, Alche´ LE (2008) The in vitro immunomodulatory activity of a synthetic brassinosteroid analogue would account for the improvement of herpetic stromal keratitis in mice. J Ster Biochem Mol Biol 108:164–170 Michelini FM, Zorrilla P, Robello C, Alche´ LE (2013) Immunomodulatory activity of an anti-HSV-1 synthetic stigmastane analog. Bioorg Med Chem 21(2):560–568 Mitchell JW, Mandava N, Worley JF, Plimmer JR, Smith MV (1970) Brassins a new family of plant hormones from rape pollen. Nature 225:1065–1066 Mitchell JW, Mandava N, Worley JF, Drowne ME (1971) Fatty hormones in pollen and immature seeds of bean. J Agric Food Chem 19:391–393 Mori K, Sakakibara M, Okada K (1984) Synthesis of naturally occurring brassinosteroids employing cleavage of 23,34epoxides as key reactions. Synthesis of brassinolide, castasterone, dolicholide, dolichosterone, homodolicholide, homodolichosterone, 6-deoxocastasterone and 6-deoxodolichosterone. Tetrahedron 40:1767–1781 Mu¨ssig C (2005) Brassinosteroid-promoted growth. Plant Biol. 7:110–117 Nakashita H, Yasuda M, Nitta T, Asami T, Fujioka S et al (2003) Brassinosteroid functions in a broad range of disease resistance in tobacco and rice. Plant J. 33: 887–898 Nakaya M, Tsukaya H, Murakami N, Kato M (2002) Brassinosteroids control the proliferation of leaf cells of Arabidopsis Thaliana. Plant Cell Physiol 43:239–244 Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70(3):461–477 Nomura T, Sato T, Bishop GJ, Kamiya Y, Yokota T (2001) Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis. Phytochemistry 57:171–178

Author's personal copy Phytochem Rev (2015) 14:1053–1072 Nomura T, Jager C, Kitasaka Y, Takeuchi K et al (2004) Brassinosteroid deficiency due to truncated steroid 5a-reductase causes dwarfism in the lk mutant of pea. Plant Physiol 135:2220–2229 Nomura T, Ueno M, Yamada Y, Takatsuto S et al (2007) Roles of brassinosteroids and related m RNAs in pea seed growth and germination. Plant Physiol 143:1680–1688 Obakan P, Arisan ED, Calcabrini A, Agostinelli E, Bolkent S, Palavan-Unsal N (2014a) Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines. Amino Acids 46(3):553–564 Obakan P, Arisan ED, Coker-Gurkan A, Palavan-Unsal N (2014b) Epibrassinolide-induced apoptosis regardless of p53 expression via activating polyamine catabolic machinery, a common target for androgen sensitive and insensitive prostate cancer cells. Prostate 74(16):1622– 1633 Oh MH, Sun J, Oh DH, Zielinski RE, Clouse SD, Huber SC (2011) Enhancing Arabidopsis leaf growth by engineering the BRASSINOSTEROID INSENSITIVE1 receptor kinase. Plant Physiol 157:120–131 Ohnishi T, Szatmari AM, Watanabe B, Fujita S et al (2006) C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis. Plant Cell 18:3275–3288 Ono E, Orika E, Nakamura T et al (2000) Application of brassinosteroid to Tabebuia alba (Bignoniaceae) plants. Rev Brasil Fisiol Vegetal 12:187–194 Parl FF (2000) Estrogens, estrogen receptor and breast cancer. In: Biomedical and health research. IOS Press, Amsterdam Pietras RJ, Weinberg OK (2005) Antiangiogenic steroids in human cancer therapy. Evid Based Complement Alternat Med 2(1):49–57 Ramı´rez JA, Teme Centurioń OM, Gros EG, Galagovsky LR (2000) Synthesis and bioactivity evaluation of brassinosteroid analogs. Steroids 65:329–337 Ra´rova´ L, Zahler S, Liebl J, Krysˇtof V, Sedla´k D, Bartu˚neˇk P, Strnad M (2012) Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells. Steroids 77:1502–1509 Roddick JG, Ikekawa N (1992) Modification of root and shoot development in monocotyledon and dicotyledon seedlings by 24-epibrassinolide. J Plant Physiol 140:70–74 Sairam RK (1994) Effects of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture stress conditions of wheat variety. Plant Growth Regul 14(2):173–181 Sakakibara M, Mori K (1982) Facile synthesis of (22R,23R)homobrassinolide. Agric Biol Chem 46:2769–2779 Sakakibara M, Mori K (1983) Improved synthesis of brassinolide. Agric Biol Chem 47:663–664 Sakakibara M, Okada K, Ichikawa Y, Mori K (1982) Synthesis of brassinolide, a plant growth promoting steroidal lactone. Heterocycles 17:301–304 Sasse JM (2003) Physiological actions of Brassinosteroids: an update. J Plant Growth Regul 22(4):276–288 Savaldi-Goldstein S, Peto C, Chory J (2007) The epidermis both drives and restricts plant shoot growth. Nature 446:199–202 Shahin SA, Sunil Kumar GB, Khan M, Doohan FM (2013) Brassinosteroid Enhances resistance to fusarium diseases of barley. Phytopathology 103:1260–1267

1071 Shen ZW, Zhou WS (1990) Study on the syntheses of brassinolide and related compounds. Part 14. Highly stereoselective construction of the side-chain of brassinosteroids utilizing the b-Alkylative 1,3-carbonyl transposition of the steroidal 22-En-24-one. J Chem Soc Perkin Trans 1: 1765–1767 Singh AP, Savaldi-Goldstein S (2015) Growth control: brassinosteroid activity gets context. J Exp Botany 66:1123– 1132 Steigerova´ J, Oklesˇt’kova´ J, Levkova´ M, Ra´rova´ L, Kola´rˇ Z, Strnad M (2010) Brassinosteroids cause cell cycle arrest and apoptosis of human breast cancer cells. Chem Biol Interact 5:487–496 Steigerova´ J, Ra´rova´ L, Oklesˇt’kova´ J, Krˇ´ızˇova´ K, Levkova´ M, Sˇvaćhova´ M, Kola´rˇ Z, Strnad M (2012) Mechanisms of natural brassinosteroid-induced apoptosis of prostate cancer cells. Food Chem Toxicol 50:4068–4076 Sun S, Chen D, Li X, Qiao S, Shi C, Li C, Shen H, Wang X (2015) Brassinosteroid signaling regulates leaf erectness in oryza sativa via the control of a specific U-type cyclin and cell proliferation. Dev Cell 34(2):220–228 Symons GM, Davies C, Shavrukov Y, Dry IB, Reid JB, Thomas MR (2006) Grapes on steroids. Brassinosteroids are involved in grape berry ripening. Plant Physiol 140:150– 158 Szekeres M, Nemeth K, Knocz-Kalman Z et al (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85:171–182 Takatsuto S, Ikekawa N (1984) Short-step synthesis of plant growth-promoting brassinosteroids. Chem Pharm Bull 32: 2001–2004 Takatsuto S, Yazawa N, Ishiguro M, Morisaki M, Ikekawa N (1984) Stereoselective synthesis of plant growth-promoting steroids, brassinolide, castasterone, typhasterol, and their 28-Nor analogues. J Chem Soc Perkin Trans 1:139–146 Takeuchi Y, Omigawa Y, Ogasawara M, Yoneyama K et al (1995) Effects of brassinosteroids on conditioning and germination of clover broomrape seeds. Plan Growth Regul 16:153–160 Tanaka K, Nakamura Y, Asami T, Yoshida S, Matsuo T, Okamoto S (2003) Physiological roles of brassinosteroids in early growth of Arabidopsis: brassinosteroids have a synergistic relationship with gibberellin as well as auxin in light-grown hypocotyl elongation. J Plant Growth Regul 22:259–271 Thompson MJ, Mandava N, Flippen-Anderson JL, Worley JF, Dutky SR, Robbins WE, Lusby W (1979) Synthesis of brassino steroids: new plant-growth-promoting steroids. J Org Chem 44:5002–5004 Tong H, Xiao Y, Liu D, Gao S, Liu L, Yin Y, Jin Y, Qian Q, Chu C (2014) Brassinosteroid regulates cell elongation by modulating gibberellin metabolism in rice. Plant Cell 26(11):4376–4393 Tsubuki M, Keino K, Honda T (1992) Stereoselective Synthesis of plant-growth-regulating steroids: brassinolide, castasterone, and their 24,25-substituted analogues. J Chem Soc Perkin Trans 1:2643–2649 Unterholzner SJ, Rozhon W, Papacek M, Ciomas J, Lange T, Kugler KG, Mayer KF, Sieberer T, Poppenberger B (2015)

123

Author's personal copy 1072 Brassinosteroids are master regulators of gibberellin biosynthesis in Arabidopsis. Plant Cell 27(8):2261–2272 Vardhini BV, Rao SSR (2002) Acceleration of ripening of tomato pericarp discs by brassinosteroids. Phytochemistry 61:843–847 Vogler F, Schmalzl C, Englhart M, Bircheneder M, Sprunck S (2014) Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod 27(3):153–167 Vragovic´ K, Sela A, Friedlander-Shani L, Fridman Y, Hacham Y, Holland N, Bartom E, Mockler TC, Savaldi-Goldstein S (2015) Translatome analyses capture of opposing tissuespecific brassinosteroid signals orchestrating root meristem differentiation. Proc Natl Acad Sci U S A 112(3):923–928 Vriet C, Russinova E, Reuzeau C (2012) Boosting crop yields with plant steroids. Plant Cell 24(3):842–857 Wachsman MB, Castilla V (2012) Antiviral properties of brassinosteroids. In: Pereira-Netto AB (ed) Brassinosteroids: practical applications in agriculture and human health. Bentham Science Publishers, Sharjah, pp 57–71 Wachsman MB, Lopez EMF, Ramirez JA, Galagovsky LR, Coto CE (2000) Antiviral effect of brassinosteroids against herpes virus and arenaviruses. Antivir Chem Chemother 11:71–77 Wachsman MB, Ramirez JA, Galagovsky LR, Cotto CE (2002) Antiviral activity of brassinosteriods derivatives against measles virus in cell cultures. Antiviral Chem Chemother 13:61–66 Wachsman MB, Castilla V, Talarico LB, Ramirez JA, Galagovsky LR, Coto CE (2004) Antiherpetic mode of action of (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one in vitro. Int J Antimicrob Agents 23(5): 524–526 Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410:380–383 Wu YD, Lou YJ (2007) Brassinolide, a plant sterol from pollen of Brassica napus L., induces apoptosis in human prostate cancer PC-3 cells. Pharmazie 62:392–395

123

Phytochem Rev (2015) 14:1053–1072 Wu CY, Trieu A, Radhakrishnan P, Kwok SF, Harris S, Zhang K, Wang J, Wan J, Zhai H, Takatsuto S, Matsumoto S, Fujioka S, Feldmann KA, Pennell RI (2008) Brassinosteroids regulate grain filling in rice. Plant Cell 2008: 2130–2145 Yamaguchi T, Wakizuka T, Hirai K, Fujii S, Fujita A (1987) Stimulation of germination in aged rice seeds by pretreatment with brassinolide. Proc Plant Growth regul Soc Am 14:26–27 Yamamato R, Demura T, Fujuda H (1997) Brassinosteroid induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol 38:980–983 Yang CJ, Zhang C, Lu YN, Jin JQ, Wang XL (2011) The mechanisms of brassinosteroids action: from signal transduction to plant development. Mol Plant 4:588–600 Ye Q, Zhu W, Li L, Zhang S, Yin Y, Ma H, Wang X (2010) Brassinosteroids control male fertility by regulating the expression of key genes involved in Arabidopsis anther and pollen development. Proc Natl Acad Sci 107(13): 6100–6105 Yokota T, Sato T, Takeuchi Y, Nomura T et al (2001) Roots and shoots of tomato produce 6-deoxo-28norcathasterone, 6-deoxo-28-nortyphasterole and 6-deoxo-28-norcastasterone, possible precursors of 28-norcastasterone. Phytochem. 58:233–238 Zhang DW, Deng XG, Fu FQ, Lin HH (2015) Induction of plant virus defense response by brassinosteroids and brassinosteroid signalling in Arabidopsis Thaliana. Planta 241: 875–885 Zhiponova MK, Vanhoutte I, Bouldof V et al (2013) Brassinosteroid production and signalling differentially control cell division and expansion in the leaf. New Phytol 197:490–502 Zhou WS, Shen ZW (1991) Study on the synthesis of brassinolide and related compounds. Part 15. Formal synthesis of brassinolide via stereoselective sulphenate-sulphoxide transformation. J Chem Soc Perkin Trans 1:2827–2830